Our long-term objectives are to define the pathway by which two haploid yeast cells fuse to become one diploid cell. Related to fertilization, conjugation is a fundamental process common to all sexually reproducing organisms. Conjugation also has close parallels to cell fusion events during development. We propose to continue our analysis of genes required for two major steps in conjugation, cell fusion and nuclear envelope fusion. Many of the genes required for cell and nuclear fusion have homologs in all eukaryotic organisms and their study will provide important clues to human cell biology, fertility and disease. During development of multicellular organisms, cells transition from a proliferative state, devoted to cell division and lacking specialized functions, to a differentiated state, in which cell division ceases and specialized cell functions are expressed. Proliferation and differentiation are mutually exclusive states, and orderly development requires that cells shut down mitotic functions as they turn on the specialized functions related to differentiation. Indeed one hallmark of cancer is that cells tend to lose differentiated functions as they re- acquire the capacity for unrestrained proliferation. Thus the coordination of mitosis and differentiation is of vital importance. Similarly, when yeast cells conjugate, they must exit the cell cycle and express proteins required for cell and nuclear fusion. However, because gene expression begins before the completion of the previous cell cycle, and because several proteins required for conjugation have other mitotic functions, yeast cells face the additional challenge of having to prevent premature activation of conjugation. The major goal of this project is to identify the specific effects of premature activation of mating functions, and identify the genes/proteins that are toxic when prematurely activated. As a specific example, we aim to understand the controls governing a key regulator of cell fusion, Fus2p, whose localization is under extraordinarily complex regulation by both the cell cycle and conjugation. Fus2p's regulation therefore serves as a central paradigm for the transition between mitosis and conjugation. We hypothesize that the regulation of Fus2p localization prevents interference with cell-cycle completion, which we will test this by identifying the downstream pathways regulated by Fus2p. We will examine the behavior of other key proteins co-opted during mating to determine if regulated localization is a general mechanism to prevent cell-cycle interference,. At the culmination of conjugation, the two nuclear envelopes fuse to create a single diploid nucleus. Because the nuclear envelope is composed of two membranes, two distinct fusion events occur in a coordinated fashion, and fusion of the inner membranes must be catalyzed by as yet unknown proteins. We hypothesize that Kar5p, a novel conjugation-induced protein, couples the inner and outer nuclear envelopes during fusion and facilitates inner-membrane fusion. Nuclear envelope fusion may be excellent paradigm for ER remodeling, an example of a critical mitotic process co-opted to serve a different function during conjugation. Public Health Relevance: As organisms grow and develop their cells transition from proliferation, when they are dividing, but lack specialized functions, to differentiation, when cell division stops and they acquire specialized functions. Successful development requires that cells not turn on the specialized functions while they are trying divide; one hallmark of cancer is that cells lose their specialized functions as they regain the capacity for unrestrained division. This project addresses the same problem in a model organism, baker's yeast, which carefully regulates the transition from cell division to being able to mate, using genes similar to human genes with relevance to human cell biology, fertility and disease.